The enhanced performance described above for EuCl-OFX was also observed against P. aeruginosa FQ-R2 (data not shown), exhibiting a bactericidal effect at sub-MIC ofloxacin concentrations in the early hours of the experiment. Eradication was achieved with EuCl-OFX at 2048 μg mL−1 (8 × MIC ofloxacin for
P. aeruginosa FQ-R2) within the first hour of assay. After brief exposure to EuCl-OFX, the zeta potential of P. aeruginosa FQ-R1 was modified in value and sign (from −26.8 to 14.5 mV). The cationic nature of Eudragit is the key factor contributing to its interaction with the negatively charged microbial cell surface. The binding neutralizes see more and even reverse the surface charge of the bacteria. At this stage, the change is reversible. Cultures under the action of OFX showed no effect, in agreement with that previously reported for Escherichia coli with ciprofloxacin (Dealler, 1991). Most of the cells treated with EuCl-OFX for 3 h revealed alterations in their shape, cytoplasmic density and irregularities in bacterial cell wall which could affect the functionality of Selleckchem GS1101 the normal cell membrane (Fig. 2a). Although ofloxacin-treated cells showed slight changes in cytoplasmic electrodensity (*, Fig. 2b), the bacterial membranes were still unaltered and cell morphology was preserved. Untreated controls show normal appearance (Fig. 2d). Exposure of P. aeruginosa
FQ-R1 to EuCl-OFX before adding detergent or lysozyme resulted in lysis of 5.6 ± 6.8% of cells (data CYTH4 not shown). Similarly, treatment with polymyxin
B resulted in lysis of 8.5 ± 4.6% of cells. Bacteria culture was weakly sensitized by EuCl-OFX to Triton X-100 and lysozyme, but strongly sensitized to SDS (Table 2). Bacteria cell lysis by lytic agents following polymyxin treatment, a known OM-disorganizing agent, did not differ significantly. By contrast, cultures treated with ofloxacin did not differ with the control. DiBAC4 is fluorescent probe voltage sensitivity that enters depolarized cells (Müeller & Straüber, 2010), used to estimate damage of membrane potential in P. aeruginosa treated with EuCl-OFX. Figure 3 presents the effects of increasing concentrations of EuCl-OFX, drug-free polymer (EuCl) and free ofloxacin on the membrane potential for three isolates of P. aeruginosa. The negative controls showed the minimum relative fluorescence intensity (Fig. 3a, e and i). Accordingly, we considered the M1 range to be undamaged cells showing no significant depolarization of cytoplasmic membrane, and the M2 range to be damaged cells. The cell proportions exhibiting dye-associated fluorescence (M2) are expressed as percentages. The results indicate a rapid depolarization of cells treated with EuCl-OFX. After 1 h exposure, DiBAC4-associated fluorescence increases in intensity between 1 and 3 log orders, depending on the concentration and the strain analyzed.